The long-term goal of this project is to understand the molecular mechanism of human mismatch repair (MMR) and its impact on human health and disease. The importance of MMR is underscored by the fact that defects in MMR genes lead to severe genomic instability and eventually to cancer predisposition, including hereditary non-polyposis colorectal cancer (HNPCC) and certain types of sporadic cancers. MMR in human cells is nick-directed and involves at least nine proteins, which are MutS alpha, MutS beta, MutL alpha, EXO1, polymerase delta, PCNA, RFC, RPA, and HMGB1. Despite extensive investigations on the initiation of mismatch-provoked excision, which appears to require MutS alpha, PCNA, RPA, and EXO1. However, how mismatch recognition by MutS proteins leads to a strand-specific excision at a single-stranded break several hundred base pairs away from the mismatch is not understood. Three current models on mismatch- provoked excision are quite controversial in terms of how many MutS molecules are required and how the strand discrimination signal is recognized. Recently, mismatch-provoked excision has been reconstituted. Interestingly, EXO1, a 5'?>3'exonuclease, was reported to be capable of bi-directional removal of mismatch;and EXO1-catalyzed 5'excision is independent of PCNA. Given the importance of PCNA in mismatch- provoked excision and the involvement of four exonucleases in the E. coli methyl-direct MMR, we hypothesize that multiple nucleases are involved in the human reaction. Indeed, we have recently identified a novel 5'?>3'mismatch excision pathway, which depends on at least MutS alpha, PCNA, a novel 5'nuclease, and a stimulating factor. This application proposes to purify and characterize both the novel 5'nuclease and the excision stimulating factor, and eventually reconstitute the PCNA-dependent 5'excision reaction in vitro using purified proteins. The purified system will then be used to evaluate three controversial models to elucidate the molecular mechanism of mismatch-provoked excision. Since defects in mismatch excision genes, e.g., the EXO1 gene, are associated with cancer development, identifying the components required for the novel 5'excision pathway will provide new diagnostic markers for HNPCC and other MMR deficient cancer syndromes.